酪胺酸亞硫酸基轉移酶之研究平台開發與其酵素催化機制之探討

Abstract

酪氨酸亞硫酸化屬於一種在生物體中常見的蛋白質轉譯後修飾，主要利用酪氨酸亞硫酸基轉移酶將3’磷酸-5’磷酸亞硫酸腺苷的亞硫酸基轉移至特定胜肽的酪氨酸的氫氧基上，蛋白質透過這樣化學性修飾除了可以調控酵素的催化特性，在許多重要的生理與病理反應中扮演了重要角色，如：發炎反應、趨化細胞激素引發的免疫反應、以及病毒入侵等。在此論文中，希望藉由發展即時偵測的生化工具有系統性地探討酪氨酸亞硫酸化的酵素機制與生理功能。首先我們利用原核表現系統成功純化出具有酵素活性且高純度之果蠅酪氨酸亞硫酸基轉移酶(Drosophila melanogaster Tyrosylprotein sulfotransferase, DmTPST)，而且建立了一套TPST之螢光檢測法。此方法有別於放射性同位素標定與質譜儀的檢測方式，是屬於連續性的螢光檢測法。檢測系統的主旨是利用重組的酚亞硫酸基轉移酶(phenol sulfotransferase, PST)催化受質 4-methylumbelliferyl sulfate (MUS) 做為亞硫酸基提供者並轉移至3’-phosphoadenosine 5’-phosphate (PAP)再生 3’-phosphoadenosine 5’-phosphosulfate (PAPS)，此核苷酸的亞硫酸基再由TPST催化。在設計的環境中，重組的 PST 不會催化 TPST 的受質，而且反應後產生 4-methylumbelliferone (MU)之螢光變化不但可以準確定量 TPST 的活性及亞硫酸化蛋白質的總量，也使TPST以蛋白質為受質進行催化。另外，先前文獻指出，在TPST上的單點突變（在果蠅中此胺基酸在酵素的位置為H269Q）將會導致甲狀腺低下所引發的侏儒症。希望利用此螢光法來研究His-269如何影響TPST的催化機制。藉由此平台不僅可以發現更多TPST的催化機制，更可以提供酵素的相關資訊，以利於未來設計藥物來調控TPST所引發的生理與病理反應。

Tyrosine O-sulfation is one of common post-translational modifications in vivo. Tyrosylprotein sulfotransferases (TPST, EC 2.8.2.20) catalyzed this reaction, which transfers a moiety of sulfuryl group from 3’-phosphoadenosine 5’-phosphosulfate (PAPS) to the hydroxyl group of specific tyrosine residues within polypeptides. Chemical modification of a protein regulates many important physiological and pathological functions including inflammation, chemokine signaling, and virus entry. In this study, I developed feasible biochemical tools to systematically study the catalytic mechanism of Drosophila melanogaster Tyrosylprotein sulfotransferase (DmTPST) and biological functions. Firstly, a heterologous TPST expression system and purification procedure was developed to produce the enzymatically active recombinant TPSTs at near homogeneity. This method is different from the radioactive isotope labeling and mass spectrometry to detect the activity of TPST. I developed the first continuous fluorometric assay for TPST. It utilized PAPS regenerated from 3’-phosphoadenosine 5’-phosphate (PAP) by a recombinant phenol sulfotransferase (PST) using 4-methylumbelliferyl sulfate (MUS) as a sulfuryl group donor. The change of fluorescence intensity of 4-methylumbelliferone (MU) corresponded directly to the amount of active TPST. This method can use a protein as a substrate study substrate specificity of TPST, relatively, radioactive isotope labeling and mass spectrometry were limited to use peptides for substrate specificity of TPST. A single point mutation, H269Q of DmTPST which resulted in dwarfism-associated hypothyroidism, would contribute to weak enzyme activity. Detailed mechanism regarding structure-function relationship was discussed. This research provides feasible biochemical tools to uncover the enzymatic mechanism of TPST and the significance of protein tyrosine sulfation. The information is conducive to design drugs for controlling physiological and pathological responses induced by TPST.